Congenital cardiovascular malformations are among the most common congenital birth defects, occurring in almost 1% of the population. However, we do not understand the underlying molecular nature of the majority of these defects. In order to develop effective in vitro stem cell and regenerative therapies aimed at preventing birth defects and healing cardiovascular diseases in adults, it is critical to have a precise understanding of the mechanisms directing cardiovascular development in vivo. Therefore, the long-term goal of our lab is to understand the mechanisms of cardiovascular specification during vertebrate development. The specific aims of this grant are to elucidate the mechanisms by which chicken ovalbumin upstream promoting-transcription factors (Coup-tfs) are required to restrict ventricular specification and promote hemangioblast specification using zebrafish. In humans, Coup-tfs are required for normal heart development. Studies in mice have demonstrated that Coup-tf2 is required for proper early cardiovascular development, but the mechanisms underlying Coup-tf2 function in these early defects are not understood. Our preliminary results suggest that zebrafish Coup-tf1a has cardiovascular defects reminiscent of mammalian Coup-tf2. Moreover, our preliminary results support the hypothesis that zebrafish Coup-tf1a has distinct requirements restricting ventricular cell specification and promoting hemangioblast specification. In Specific Aim 1, we will use cell transplantation and lineage tracing experiments to determine which cells required Coup-tf1a to restrict ventricular cell specification. We will also determine the genetic relationship of FGF8 and Coup-tf1a in restricting ventricular cell specification. A role for Coup-tfs in vertebrate hemangioblast specification has not been previously recognized. In Specific Aim 2, we will use epistasis and cell transplantation analysis to determine the relationship of Coup-tf1a to known regulators of hemangioblast specification and what cells require Coup-tf1a for hemangioblast specification. Factors downstream of Coup-tf1a in ventricular cell and hemangioblast specification are not known. In Specific Aim 3, we will use loss-of-function methods to determine if candidate genes are required for regulating these distinct processes downstream of Coup-tf1a. Altogether, these studies will improve our understanding of normal cardiovascular specification events during development, providing the basis for future therapies aimed at healing congenital cardiovascular defects in children and injured or diseased cardiovascular tissues in adults.